A variety of genetic abnormalities arise in human cancer that contribute to neoplastic transformation and malignancy. Instability of the genome generates mutations that alter cell proliferation, angiogenesis, metastasis, and tumor immunogenicity. Despite a better understanding of the molecular basis of cancer, many malignancies remain resistant to traditional forms of treatment. The definition of tumor-associated genetic mutations, however, has heightened interest in cancer as a target for gene therapy. Immunotherapy has shown promise as a primary approach to the treatment of malignancy. Indeed, specific cancers, such as melanoma or renal cell carcinoma, are relatively more responsive to modulation of immune function, possibly because the immune system can be induced to recognize mutant gene products in these cells. Conventionally, approaches to immunotherapy have involved the administration of non-specific immunomodulating agents such as Bacillus Calmette-Guerin (BCG), cytokines, and/or adoptive T cell transfer, which have shown promise in animal models (B. Zbar, et al., J. Natl. Canc. Inst. 46, 831 (1971); S. A. Rosenberg, et al., J. Exp. Med. 16, 1169 (1985); S. Shu, and S. A. Rosenberg, Cancer Res. 45, 1657 (1985); P. J. Spiess, et al., J. Natl. Canc. Inst. 79, 1067; T. Chou, et al., J. Immunol. 140, 2453 (1988); H. Yoshizawa, et al., J. Immunol. 147, 729 (1991)) and in man (D. L. Morton, et al., Ann. Surg. 180, 635 (1974); S. A. Rosenberg, et al., Ann. Surg. 210, 474 (1989); S. A. Rosenberg, et al., N. Eng. J. Med. 319, 1676 (1988); R. L. Kradin, et al., Lancet 577 (1989)). More recently, molecular genetic interventions have been designed in an attempt to improve the efficacy of immunotherapy. Human gene transfer protocols have been designed to monitor the traffic of lymphocytes into melanoma tumors (S. A. Rosenberg, et al., N. Eng. J. Med. 323, 570 (1990)) or to introduce cytokine genes into tumor cells to stimulate the host's immune response to residual tumor (S. A. Rosenberg, Hum. Gene Ther. 3, 57 (1992)).
Recently, a new molecular genetic intervention has been developed for human malignancy. This approach relies on the direct transmission of recombinant genes into established tumors in vivo to genetically modify them as they grow in situ. In animal models, introduction of a gene encoding a foreign major histocompatibility (MHC) protein (class I) in vivo signals the immune system to respond to the foreign antigen (G. E. Plautz, et al., Proc. Natl. Acad. Sci. USA 90, 4645 (1993); E. G. Nabel, et al., Proc. Natl. Acad. Sci. USA 89, 5157 (1992)). More importantly, when this gene is transduced into established tumors in vivo, a cytolytic T cell response is also generated against unmodified tumor cells. In murine models, this approach has led to significant reductions in tumor growth and, in some cases, complete remission (G. E. Plautz, et al., Proc. Natl. Acad. Sci. USA 90, 4645 (1993)). Based on these studies, approval was recently received from the Recombinant DNA Advisory Committee of the National Institutes of Health to conduct a human clinical protocol using direct transfer of a human transplantation antigen gene in an effort to treat malignancy. This protocol proposed to perform direct gene transfer in humans and to utilize a non-viral vector which reduces several safety concerns about viral vectors. This clinical trial involved the treatment of patients with metastatic melanoma at subcutaneous lesions. The treatment constituted intratumoral injection of the human class I MHC gene, HLA-B7, complexed to a cationic liposome, DC-Cholesterol (G. J. Nabel, Hum. Gene Ther. 3, 705 (1992); X. Gao and L. Huang, Biochem. Biophys. Res. Commun. 179, 280 (1991)). These patients received escalating doses of the DNA liposome complex. Recombinant gene expression, toxicity, and the immunologic response to treatment is being evaluated. Based on animal studies, no toxicities had been readily apparent using these modes of direct gene transfer in vivo in short-term or long-term studies (G. J. Nabel, Hum. Gene Ther. 3, 399 (1992); G. J. Nabel, Hum. Gene Ther. 3, 705 (1992); M. J. Stewart, et al., Hum. Gene Ther. 3, 267 (1992)). Taken together, these studies were intended to determine whether direct gene transfer was an appropriate form of treatment for malignancy.
Direct Gene Transfer and Modulation of the Immune System
The utilization of catheter-based gene delivery in vivo provided a model system for the introduction of recombinant gene-containing molecules into specific sites in vivo. Early studies focused on the demonstration that specific reporter genes could be expressed in vivo (E. G. Nabel, et al., Science 249, 1285 (1990); E. G. Nabel, et al., Science 244, 1342 (1989)). Subsequent studies were designed to determine whether specific biologic responses could be induced at sites of recombinant gene transfer. To address this question, a highly immunogenic molecule, a foreign major histocompatibility complex (MHC), was used to elicit an immune response in the iliofemoral artery using a porcine model. The human HLA-B7 gene was introduced using direct gene transfer with a retroviral vector or DNA liposome complex (E. G. Nabel, et al., Proc. Natl. Acad. Sci. USA 89, 5157 (1992)). With either delivery system, expression of the recombinant HLA-B7 gene product could be demonstrated at specific sites within the vessel wall. More importantly, the expression of this foreign histocompatibility antigen induced an immunologic response at the sites of genetic modification. This response included a granulomatous mononuclear cell infiltrate beginning 10 days after introduction of the recombinant gene. This response resolved by 75 days after gene transfer; however, a specific cytolytic T cell response against the HLA-B7 molecule was persistent. This study demonstrated that a specific immunologic response could be induced by the introduction of a foreign recombinant gene at a specific site in vivo. Moreover, this study provided one of the first indications that direct gene transfer of specific recombinant genes could elicit an immune response to the product of that gene in vivo (E. G. Nabel, et al., Proc. Natl. Acad. Sci. USA 89, 5157 (1992)).
These studies suggested that the introduction of the appropriate recombinant genes could be used to stimulate the immune system to recognize its product in vivo. In addition, this approach provided a general method for the induction of a specific site in vivo. To determine whether direct gene transfer might be appropriate for the treatment of disease, a murine model of malignancy was developed. Direct gene transfer of an allogeneic histocompatibility complex gene into a murine tumor was found to elicit an immune response not only to the foreign MHC protein but also to previously unrecognized tumor-associated antigens. These immune responses were T cell-dependent, and these tumor-associated proteins were recognized within the context of the self major histocompatibility complex. In animals presensitized to a specific MHC haplotype, direct gene transfer into established tumors could attenuate tumor growth or, in some cases, lead to complete tumor regression (G. E. Plautz, et al., Proc. Natl. Acad. Sci. USA 90, 4645 (1993)). These studies demonstrated that direct gene transfer of foreign MHC genes into tumors have potentially therapeutic effects that may be appropriate for the treatment of malignancy.
Immunotherapy of Malignancy
In some instances, the immune system appears to contribute to the surveillance and destruction of neoplastic cells, by mobilization of either cellular or humoral immune effectors. Cellular mediators of anti-tumor activity include MHC-restricted cytotoxic T cells, natural killer (NK) cells (R. K. Oldham, Canc. Metast. Rev. 2, 323 (1983); R. B. Herberman, Concepts Immunopathol. 1, 96 (1985)) and lymphokine-activated killer (LAK) cells (S. A. Rosenberg, Immunol. Today 9, 58 (1988)). Cytolytic T cells which infiltrate tumors have been isolated and characterized (I. Yron, et al., J. Immunol. 125, 238 (1980)). These tumor infiltrating lymphocytes (TIL) selectively lyse cells of the tumor from which they were derived (P. J. Spiess, et al., J. Natl. Canc. Inst. 79, 1067; S. A. Rosenberg, et al., Science 223, 1318 (1986)). Macrophages can also kill neoplastic cells through antibody-dependent mechanisms (J. Marcelletti and P. Furmanski, J. Immunol. 120, 1 (1978); P. Ralph, et al., J. Exp. Med. 167, 712 (1988)), or by activation induced by substances such as BCG (P. Alexander, Natl. Cancer Inst. Monogr. 39, 127 (1973)).
Cytokines can also participate in the anti-tumor response, either by a direct action on cell growth or by activating cellular immunity. The cytostatic effects of tumor necrosis factor-.alpha. (TNF-.alpha.) (L. J. Old, Science 230, 630 (1985)) and lymphotoxin (M. B. Powell, et al., Lymphokin Res. 4, 13 (1985)) can result in neoplastic cell death. Interferon-.gamma. (IFN-.gamma.) markedly increases class I MHC cell surface expression (P. Lindahl, et al., Proc. Natl. Acad. Sci. USA 70, 2785 (1973); P. Lindahl, et al., Proc. Natl. Acad. Sci. USA 73, 1284 (1976)) and synergizes with TNF-.alpha. in producing this effect (L. J. Old, Nature 326, 330 (1987)). Colony stimulating factors such as G-CSF and GM-CSF activate neutrophils and macrophages to lyse tumor cells directly (S. C. Clark and R. Kamen, Science 236, 1229 (1987)), and interleukin-2 (IL-2) activates Leu-19+ NK cells to generate lymphokine activated killer cells (LAK) capable of lysing autologous, syngeneic or allogeneic tumor cells but not normal cells (S. A. Rosenberg, Immunol. Today 9, 58 (1988); M. T. Lotze, et al., Cancer Res. 41, 4420 (1981); C. S. Johnson, et al., Cancer Res. 50, 5682 (1990)). The LAK cells lyse tumor cells without preimmunization or MHC restriction (J. H. Phillips and L. L. Lanier, J. Exp. Med. 164, 814 (1986)). Interleukin-4 (IL-4) also generates LAK cells and acts synergistically with IL-2 in the generation of tumor specific killers cells (J. J. Mule, et al., J. Immunol. 142, 726 (1989)).
Since most malignancies arise in immunocompetent hosts, it is likely that tumor cells have evolved mechanisms to escape host defenses, perhaps through evolution of successively less immunogenic clones (G. Klein and E. Klein, Proc. Natl. Acad. Sci. USA 74, 2121 (1977)). Several studies suggest that reduced expression of MHC molecules may provide a mechanism to escape detection by the immune system. Normally, the class I MHC glycoprotein is highly expressed on a wide variety of tissues and, in association with .beta.-2 microglobulin, presents endogenously synthesized peptide fragments to CD8 positive T cells through specific interactions with the CD8/T-cell receptor complex (P. J. Bjorkman and P. Parham, Ann. Rev. Biochem. 59, 253 (1990). Deficient expression of class I MHC molecules could limit the ability of tumor cells to present antigens to cytotoxic T cells. Freshly isolated cells from naturally occurring tumors frequently lack class I MHC antigen completely or show decreased expression (C. A. Holden, et al., J. Am. Acad. Dermatol. 9, 867 (1983); N. Isakov, et al., J. Natl. Canc. Inst. 71, 139 (1983); W. Schmidt, et al., Immunogen. 14, 323 (1981); K. Funa, et al., Lab Invest. 55, 185 (1986); L. A. Lampson, et al., J. Immunol. 130, 2471 (1983)). Reduced class I MHC expression could also facilitate growth of these tumors when transplanted into syngeneic recipients. Several tumor cell lines which exhibit low levels of class I MHC proteins become less oncogenic when expression vectors encoding the relevant class I MHC antigen are introduced into them (K. Tanaka, et al., Science 228, 26 (1985); K. Hui, et al., Nature 311, 750 (1984); R. Wallich, et al., Nature 315, 301 (1985); H-G. Ljunggren and K. Karre, J. Immunogenet. 13, 141 (1986); G. J. Hammerling, et al., J. Immunogenet. 13, 153 (1986)). In some experiments, tumor cells which express a class I MHC gene confer immunity in naive recipients against the parental tumor (K. Hui and F. Grosveld, H. Festenstein, Nature 311, 750 (1984); R. Wallich, et al., Nature 315, 301 (1985)). The absolute level of class I MHC expression however, is not the only factor which influences the tumorigenicity or immunogenicity of tumor cells. In one study, mouse mammary adenocarcinoma cells, treated with 5-azacytidine and selected for elevated levels of class I MHC expression did not display altered tumorigenicity compared to the parent line (D. A. Carlow, et al., J. Natl. Canc. Inst. 81, 759 (1989)).
The immune response to tumor cells can be stimulated by systemic administration of IL-2 (M. T. Lotze, et al, J. Immunol. 135, 2865 (1985)), or IL-2 with LAK cells (S. A. Rosenberg, et al., N. Eng. J. Med. 316, 889 (1987); C. S. Johnson, et al., Leukemia 3, 91 (1989)). Clinical trials using tumor infiltrating lymphocytes are also in progress (S. A. Rosenberg, et al., N. Eng. J. Med. 323, 570 (1990)). Recently, several studies have examined the tumor suppressive effect of lymphokine production by genetically altered tumor cells. The introduction of tumor cells transfected with an IL-2 expression vector into syngeneic mice stimulated an MHC class I restricted cytolytic T lymphocyte response which protected against subsequent rechallenge with the parental tumor cell line (E. R. Fearon, et al., Cell 60, 397 (1990)). Expression of IL-4 by plasmacytoma or mammary adenocarcinoma cells induced a potent anti-tumor effect mediated byinfiltration of eosinophils and macrophages (R. I. Tepper, et al., Cell 57, 503 (1989)). These studies demonstrate that cytokines, expressed at high local concentrations, are effective anti-tumor agents.
An alternative approach has recently been proposed to stimulate an anti-tumor response through the introduction of an allogeneic class I MHC gene into established human tumors (supra). The antigenicity of tumor cells had been altered previously by the expression of viral antigens through infection of tumor cells (J. Lindenmann and P. A. Klein, J. Exp. Med. 126, 93 (1967); Y. Shimizu, et al., Eur. J. Immunol. 14, 839 (1984); H. Yamaguchi, et al., Cancer Immunol. Immunother. 12, 119 (1982); M. Hosokama, Cancer Res. 43, 2301 (1983); V. Shirrmacher and R. Heicappell, Clin. Exp. Metastasis 5, 147 (1987)), or expression of allogeneic antigens introduced by somatic cell hybridization (J. F. Watkins and L. Chen, Nature 223, 1018 (1969); N. Kuzumaki, et al., Eur. J. Cancer. 15, 1253 (1979)). Allogeneic class I MHC genes had been introduced into tumor cells by transfection and subsequent selection in vitro. These experiments produced some conflicting results. In one case, transfection of an allogeneic class I MHC gene (H-2L.sup.d) into an H-2.sup.b tumor resulted in immunologic rejection of the transduced cells and also produced transplantation resistance against the parent tumor cells (T. Itaya, et al., Cancer Res. 47, 3136 (1987)). In another instance, transfection of H-2.sup.b melanoma cells with the H-2D.sup.d gene did not lead to rejection (J. E. Talmadge, et al., Proc. Amer. Assoc. for Cancer Res. 26, 59 (1985)), however increased differential expression of H-2D products relative to H-2K may have affected the metastatic potential and immunogenicity of tumor cells (J. Gopas, et al., Adv. Cancer Res. 53, 89 (1989)). The effects of allogeneic H-2K gene expression in tumor cells was examined in another study (G. A. Cole, et al., Proc. Natl. Acad. Sci. USA 84, 8613 (1987)). Several subclones which were selected in vitro and expressed an allogeneic gene were rejected in mice syngeneic for the parental tumor line, however, other subclones did not differ from the parental, untransduced line in generating tumors. This finding suggests that clone-to-clone variation in in vivo growth and tumorigenic capacity may result in other modifications of cells, caused by transfection or the subcloning procedure, which affects their tumorigenicity. These types of clonal differences may be minimized by transducing a population of cells directly in vivo.
Gene Therapy Approaches
The immune system can provide protection against cancer and may play an important role as an adjuvant treatment for malignancy. Lymphokine activated killer cells (LAK) and tumor infiltrating lymphocytes (TIL) can lyse neoplastic cells and produce partial or complete tumor rejection. Expression of cytokine genes in malignant cells has also enhanced tumor regression. Because current strategies to stimulate an immune response against tumor cells often fail to eradicate tumors, an important goal of immunotherapy is to improve upon current techniques and understand the mechanisms of immune recognition.
A model has been described for the immunotherapy of malignancy using a gene encoding a transplantation antigen, an allogeneic class I major histocompatibility complex (MHC) antigen, introduced into human tumors in vivo by DNA/liposome transfection (G. J. Nabel, Hum. Gene Ther. 3, 399 (1992); G. J. Nabel, Hum. Gene Ther. 3, 705 (1992)). Expression of allogeneic MHC antigens on tumor cells stimulates immunity against both the allogeneic MHC gene on transduced cells as well as previously unrecognized antigens in unmodified tumor cells (G. E. Plautz, et al., Proc. Natl. Acad. Sci. USA 90, 4645 (1993)). The introduction of an allogeneic MHC gene directly into tumors in vivo has induced partial tumor regressions, as well as the specific cytotoxic T cell response to other antigens. In a recent trial in humans, no toxicity of this form of treatment was observed. It is an object of the present invention to optimize this gene therapy approach.